We are investigating the mechanisms by which the adhesive glycoproteins TSP1 and TSP2 regulate tumor growth, metastasis, and angiogenesis. Data from tumor xenograft and transgenic mouse models demonstrate that TSP1 and TSP2 are suppressors of tumor progression. Although the suppressive activity of TSP1 was initially ascribed to inhibition of angiogenesis, we have obtained evidence for significant direct effects of TSP1 on both tumor cells and the host immune response. Using synthetic peptides, recombinant TSP1 fragments, and mutagenesis, we are defining receptors and recognition sequences that mediate activities of TSP1 toward each of these cell types. We are defining the cell-specific signal transduction pathways for multiple TSP receptors using TSP1-null transgenic mice and by identifying genes that are regulated by these TSP1-initiated signals in specific cell types. We have identified peptide sequences in TSP1 that mimic the anti-angiogenic activities of the whole molecule. Stable analogs of these peptides inhibited angiogenesis in several animal models and are being developing for therapeutic applications.In addition to the three known a1 integrin recognition sites in the N-module of TSP1, we found that a1 integrins mediate cell adhesion to the type 1 and type 2 repeats. The type 1 repeats of TSP1 differ from typical integrin ligands in that recognition is pan-a1 specific. a1 integrins recognize both the second and third type 1 repeats, and each type 1 repeat shows pan-a1 specificity and divalent cation-dependence for promoting cell adhesion. a1 integrin expression is necessary for cell adhesion to the type 1 or type 2 repeats, and a1 integrins bind in a divalent cation-dependent manner to a type 1 repeat affinity column. These results provide a new mechanism for previously reported biological activities of this domain of TSP1.We have reexamined the role of endogenous TSP1 in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in 3-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet derived growth factor dependent SMC migration and/or proliferation.Redox signaling plays an important role in the positive regulation of angiogenesis by vascular endothelial growth factor, but its role in signal transduction by angiogenesis inhibitors is less clear. Using muscle explants in 3D culture, we found that explants from mice lacking TSP1 exhibit exaggerated angiogenic responses to an exogenous NO donor, which could be reversed by providing exogenous TSP1. To define the basis for inhibition by TSP1, we examined the effects of TSP1 on several pro-angiogenic responses of endothelial cells to NO. NO has a biphasic effect on endothelial cell proliferation. The positive effect at low doses of NO is sensitive to inhibition of cGMP signaling and to picomolar concentrations of TSP1. NO stimulates both directed (chemotactic) and random (chemokinetic) motility of endothelial cells in a cGMP-dependent manner. TSP1 potently inhibits chemotaxis stimulated by NO. Low doses of NO also stimulate adhesion of endothelial cells on type I collagen in a cGMP-dependent manner.